Correlation between L‐amino acid transporter 1 expression and 4‐borono‐2‐18F‐fluoro‐phenylalanine accumulation in humans

Abstract Background The correlation between L‐type amino acid transporter 1 (LAT1) expression and 4‐borono‐2‐18F‐fluoro‐phenylalanine (18F‐FBPA) accumulation in humans remains unclear. This study aimed to investigate the correlation between LAT1 expression in tumor tissues and 18F‐FBPA accumulation in patients with head and neck cancer who participated in a clinical trial of 18F‐FBPA positron emission tomography (PET). Methods Altogether, 28 patients with head and neck cancer who participated in a clinical trial of 18F‐FBPA PET at our institution between March 2012 and January 2018 were included. Correlations between standardized uptake values (SUVs); the maximum SUV (SUVmax), the mean SUV within a 1 cm3 sphere centered at a single point, that is, the SUVmax (SUVpeak), the minimum SUV (SUVmin), and the intensity of LAT1 expression (maximum and minimum LAT1 expressions) were investigated. Results Weak correlations were identified between SUVmax and LAT1 maximum score, SUVmin and LAT1 maximum score, and SUVmin and LAT1 minimum score (ρ = 0.427, 0.362, and 0.330, respectively). SUVmax and LAT1 minimum score, SUVpeak and LAT1 maximum score, and SUVpeak and LAT1 minimum score demonstrated moderate correlations (ρ = 0.535, 0.556, and 0.661, respectively). Boron neutron capture therapy (BNCT) was performed in 2 of the 4 patients with discrepancies between 18F‐FBPA accumulation and intensity of LAT1 expression, and the intensity of LAT1 expression was a better predictor of treatment response. Conclusion 18F‐FBPA accumulation and the intensity of LAT1 expression demonstrated a moderate correlation; however, LAT1 expression may be a better predictor of treatment response of BNCT in patients with discrepancies.


| INTRODUCTION
2][3][4][5] α-Rays and 7 Li recoil nuclei are high-linear energy transfer radiation with high biological effects, and their range is almost the same as the cell diameter; thus, cancer cells can be selectively destroyed.The results of a phase II clinical trial of BNCT for unresectable recurrent and locally advanced head and neck cancers 6,7 revealed a high tumor response rate of 71% with few severe side effects.Consequently, BNCT was approved in Japan in 2020 for the treatment of patients with unresectable recurrent or locally advanced head and neck cancers.Borofalan, formerly known as 4-borono-L-phenylalanine (BPA), was used as 10 B in the BNCT.Sufficient 10 B uptake by cancer cells is critical for effective BNCT.The uptake of BPA into cancer cells is mediated by L-type amino acid transporter 1 (LAT1). 8,91][12][13][14] In previous studies on glioma cells, a high selectivity of 18 F-FBPA for LAT1 expression has been reported. 8,15][18] The accumulation of 18 F-FBPA in tumor tissues has been reported to be affected by stromal cell density. 19However, by examining LAT1 expression in tumor tissues from patients, we can analyze BPA accumulation at the microscopic level and estimate the therapeutic effect of BNCT more accurately. 20n this study, the LAT1 expression in tumor tissues of patients with head and neck cancer who underwent 18 F-FBPA PET/computed tomography (CT) in clinical trials at our institution from 2012 to 2018 was investigated.The first aim of this study was to determine the correlation between 18 F-FBPA accumulation in 18 F-FBPA PET images and LAT1 expression.The second aim was to characterize the cases with discrepancies in LAT1 expression intensity and 18 F-FBPA accumulation.

| Patient population
A flowchart of the patient inclusion process is presented in Figure 1.Data of 120 patients in the clinical trial of 18 F-FBPA PET/CT at our institution between March 2012 and January 2018 were retrieved.Among these patients, those without tumor tissue or those for whom 18 F-FBPA PET images could not be analyzed because of a lack of information at the time of imaging, such as the method of 18 F-FBPA administration, were excluded.Finally, the data from 28 patients were included in the analysis.The following factors were investigated: age, sex, primary tumor sites, pathology, tissue sampling method (biopsy or surgery), gross tumor volume (GTVs), and the interval between the biopsy date and 18 F-FBPA PET/CT imaging date.

| Method for evaluating 18F-FBPA accumulation on 18F-FBPA PET/CT images
A previous study described the details of the methods of performing an 18 F-FBPA PET/CT scan. 21 18F-FBPA images were acquired 60 min after injection using a PET/ CT scanner (Discovery 600; GE Healthcare, Milwaukee).All 18 F-FBPA images were confirmed by a board-certified radiologist or nuclear physician.SUVs were measured score demonstrated moderate correlations (ρ = 0.535, 0.556, and 0.661, respectively).Boron neutron capture therapy (BNCT) was performed in 2 of the 4 patients with discrepancies between 18 F-FBPA accumulation and intensity of LAT1 expression, and the intensity of LAT1 expression was a better predictor of treatment response. 18F-FBPA accumulation and the intensity of LAT1 expression demonstrated a moderate correlation; however, LAT1 expression may be a better predictor of treatment response of BNCT in patients with discrepancies.

K E Y W O R D S
4-borono-2-18 F-fluoro-phenylalanine positron emission tomography, boron neutron capture therapy, boronophenylalanine, L-type amino acid transporter 1, neoplasm using the AW Volume Share 4.5 software (GE Healthcare, Milwaukee).The GTVs and regions of interest (ROIs) were drawn by a radiation oncologist and a board-certified radiologist/nuclear medicine technician, respectively.The maximum SUV (SUV max ) of the tumors in the ROIs was defined as the area with the highest activity.SUV max , the mean SUV within a 1 cm 3 sphere centered at a single point, that is, SUV max (SUV peak ) and the minimum SUV (SUV min ) were used to evaluate the intensity of 18 F-FBPA accumulation, and kurtosis was used to evaluate heterogeneity.

LAT1 expression
Tumor tissues removed by clinical biopsy or surgery were prepared as 4-mm-thick formalin-fixed, paraffinembedded tissue sections.The monoclonal antibody was anti-LAT1 antibody (4A2, 2 mg/mL) diluted 8000-fold, and the antigen was removed by microwave treatment at 98°C for 40 min in citrate buffer (pH 6.0, 0.01 mol/L).Immunostaining was performed using a Dako Autostainer Link 48 (Agilent Technologies).The intensity of LAT1 expression was evaluated by a pathologist using a previously reported 4-point scoring from 0 to 3, with 0 being the weakest expression and 3 being the strongest. 23Examples of scoring are demonstrated in Figure 2. The intensity of LAT1 expression was assessed using the maximum and minimum plasma membrane scores of each sample.Heterogeneity was considered present if the maximum and minimum scores differed.

| Methods for evaluating the correlations between 18F-FBPA accumulations and LAT1 expressions (statistical analyses)
Data on LAT1 expression and 18 F-FBPA accumulation were collected independently, and the correlation analysis was performed by one physician.Correlations between parameters related to 18 F-FBPA accumulation, such as SUV max and SUV peak and SUV min , and those related to the intensity of LAT1 expression based on the maximum and minimum scores were investigated.The correlations between the heterogeneity of 18 F-FBPA accumulation and LAT1 expression were also investigated.The strength of the correlations was evaluated using the Spearman's rank correlation coefficient.A correlation coefficient (ρ) of ≥0.3 was considered correlative.Furthermore, correlations of 0.3 ≤ ρ ≤ 0.5, 0.5 ≤ ρ ≤ 0.7, and 0.7 ≤ ρ were considered weak, moderate, and strong, respectively.To characterize patients with discrepancies between the intensity of LAT1 expression and 18 F-FBPA accumulation, detailed data including dynamic 18 F-FBPA PET findings were investigated regarding the patients with a maximum LAT1 score of 3 but SUV peak < 2.5 and those with a maximum LAT1 score of 0 but SUV peak ≥ 2.5.Statistical significance was set at p < 0.05.All the statistical analyses were performed using the IBM SPSS version 26 software (IBM Corp.).

| Ethical approval
All the analyses performed in this study were approved by the appropriate institutional review board (approval number: 2020-277) and were performed in accordance with the ethical standards of the committee and the 1964 Helsinki Declaration and its later amendments.Patient and treatment characteristics are summarized in Table 1.The median age was 56.5 years (range, 6-76 years).Oral cancer was the most common primary cancer.The pathological diagnosis was squamous cell carcinoma in 10 patients (35.7%) and rhabdomyosarcoma in 6 patients (21.4%).The tissue sampling method was biopsy in 12 patients (42.9%) and surgery in 16 patients (57.1%).The median volume of GTVs was 34.4 cm 3 (1.5-678.1).The LAT1 maximum scores were 0, 1, 2, and 3, and the minimum scores were 7, 5, 6, and 10 for 3, 5, 2, and 18 patients, respectively.If the sphere centered on SUV max extends beyond the GTV, the SUV peak cannot be calculated because it includes nearby normal tissue.Therefore, the SUV peak could not be calculated for six patients.The median SUV max , SUV peak , and SUV min were 3.70 (range, 1.25-12.42),3.00 (range, 1.05-10.84),and 0.53 (0.04-1.90).The median kurtosis was −0.23 (−1.06-1.51).Thirteen patients (46.4%) were classified as having a heterogeneous LAT1 expression.Treatment prior to biopsy was chemotherapy plus radiation therapy in 7 patients, chemotherapy in 2 patients, radiation therapy in 2 patients, and surgery in 1 patient; meanwhile, 16 patients were untreated.The median number of days between specimen sampling and 18 F-FBPA PET/ CT imaging was 166.5 days (range, 4-2410 days).Between specimen sampling and 18 F-FBPA PET/CT imaging, 18 patients received treatment: 9 received chemotherapy, 8 received chemotherapy plus radiation therapy, and 1 received radiation therapy.
The correlations between SUVs and LAT1 scores are presented in Table 2. Immunostained images with LAT1 antibody and 18 F-FBPA PET/CT images of the same patients are shown in Figure 3. Weak correlations were identified between SUV max and LAT1 maximum score, between SUV min and LAT1 maximum score, and between SUV min and LAT1 minimum score (ρ = 0.427, 0.362, and 0.330, respectively).SUV max and LAT1 minimum score, SUV peak and LAT1 maximum score, and SUV peak and LAT1 minimum score (Figure 4) demonstrated moderate correlations (ρ = 0.535, 0.556, and 0.661, respectively).The median kurtosis of SUVs in patients with heterogeneity in LAT1 expression was −0.35 (range, −1.06-1.14),and that in patients without heterogeneity was −0.17 (range, −1.03-1.51).No correlation was identified between the heterogeneity of LAT1 expression and the kurtosis of SUV accumulation (ρ = 0.168).
Patients with discrepancies between 18 F-FBPA accumulation and LAT1 expression intensity are summarized in Table 3.Three patients with a LAT1 score of 3 had an SUV peak < 2.5 (Table 3, patients A-C).The pathological diagnoses were adenoid cystic carcinoma, squamous cell carcinoma, and Ewing's sarcoma in one patient each.Two patients demonstrated heterogeneity in LAT1 expression levels.The median number of days between specimen sampling and 18 F-FBPA PET/CT imaging was 570 days (range 5-1423 days).The median volume of GTVs was 29.0 cm 3 (11.3-88.4).In addition, on dynamic 18 F-FBPA Abbreviations: LAT1, L-type amino acid transporter 1; SUV, standardized uptake value; SUV max , maximum SUV; SUV min , minimum SUV; SUV peak , mean SUV within a 1 cm 3 sphere centered at a single point that is the SUV max .
PET/CT, SUV max at 10 min after 18 F-FBPA administration was 5.22, 5.01, and 4.34 for patients A-C, respectively, which was washed out early and remained low at 1 h (Figure 3C).Patient C was treated with BNCT for a primary tumor in the parotid gland after laminectomy of the 12th thoracic metastasis and prior to 18 F-FBPA PET   3).(C-1) Maximum and minimum LAT1 expressions were 3 and 2, respectively.(C-2, 3) SUV max at 10 min after 18 F-FBPA administration was high (5.01,C-2), but washed out early and remained low at 1 h (2.65, C-3).
imaging; the SUV max was 3.1 for the primary tumor site and 3.0 for the 12th thoracic metastasis site.After BNCT, the tumor significantly shrank but remained in the deep part of the tumor, where neutrons could not reach.By contrast, one patient with a LAT1 score of 0 had an SUV peak ≥ 2.5 (Table 3, patient D).The pathological diagnosis was rhabdomyosarcoma.The number of days between specimen sampling and 18 F-FBPA PET/CT imaging was 821 days.The GTV was 207.9 cm 3 .In the patient, no LAT1 expression was observed on the plasma membrane, but staining was detected in the cytoplasm.The patient also underwent an 18 F-FBPA PET/CT at the time of postoperative recurrence.The patient was treated with BNCT after 18 F-FBPA PET/CT; however, the effect was poor as the tumor grew.

| DISCUSSION
In our study, we demonstrated a correlation between LAT1 expression intensity and SUV accumulation on 18 F-FBPA PET/CT images.The strongest correlation was between the LAT1 minimum score and SUV peak .The minimum score demonstrating a stronger correlation than the maximum LAT1 score may possibly be because the LAT1 score was evaluated at the microscopic level, and even if the maximum score was high, a large area with a low minimum score may not lead to 18 F-FBPA accumulation at the macroscopic level.Therefore, scoring that considers not only the intensity of expression, but also the range, such as the tumor proportion score for programmed cell death ligand 1, may be more useful in predicting the therapeutic effect of BNCT.In this study, patients with discrepancies in LAT1 expression and 18 F-FBPA accumulation were further investigated.All patients with a strong LAT1 expression demonstrated a high accumulation of 18 F-FBPA at 10 min, according to further detailed analysis of dynamic 18 F-FBPA PET, although 18 F-FBPA was washed out early.As 1-h values are commonly used for 18 F-FBPA PET measurements, 12,14,24 the SUV values were considered low.In a previous study on dynamic 18 F-FBPA PET, 18 F-FBPA uptake was reported to be washed out early in squamous cell carcinoma, 30 min after 18 F-FBPA administration. 21ecause BPA is administered continuously by intravenous infusion during BNCT, even patients whose 18 F-FBPA is F I G U R E 4 Correlation between the mean standardized uptake value (SUV) within a 1 cm 3 sphere centered at a single point that is the maximum SUV (SUV peak ) and the maximum L-type amino acid transporter 1 (LAT1) score.

T A B L E 3
Patients with a discrepancy between the intensity of 4-borono-2-18 F-fluoro-phenylalanine accumulation and L-type amino acid transporter 1 (LAT1) expression.washed out early will benefit from BNCT.Patient C had a high LAT1 expression in the surgical specimen of the 12th thoracic metastasis; however, 18 F-FBPA accumulation was low, with an SUV max of 3.0.The therapeutic effect of BNCT on primary tumors was high.In patient D, although LAT1 expression was absent, 18 F-FBPA accumulation was high, with an SUV max of 3.5.The patient was subsequently treated with BNCT, which did not result in tumor shrinkage.In these cases, LAT1 expression may be more useful in predicting the treatment response after BNCT than 18 F-FBPA PET.The small tumor volume and heterogeneity of LAT1 expression in patient A may have influenced the low 18 F-FBPA accumulation.Furthermore, a time gap was observed between specimen sampling and 18 F-FBPA PET/CT imaging, which may have changed the nature of the tumor. 18F-FBPA PET has been used to predict the therapeutic effects of BNCT using borofalan.In a previous study of 18 F-FBPA PET in rat xenograft models of C6 glioma, high selectivity of 18 F-FBPA for LAT1 was reported. 15Previous studies have identified a correlation between 18 F-FBPA accumulation on 18 F-FBPA PET by bolus injection and BPA uptake by continuous infusion in mice. 25,26Furthermore, Imahori et al. 27 reported a correlation between 18 F-FBPA accumulation on 18 F-FBPA PET and 10 B accumulation in surgical specimens of patients with high-grade gliomas.However, Aihara et al. 28 reported that the SUV max /SUV of normal tissue (SUV N ), mean SUV, SUV min , and Vo2.5 (tumor volume ratio of SUV max /SUV N ≥ 2.5) were not prognostic factors for complete response rate.In their study, SUV min /SUV N was the only significant factor; however, SUV min and SUV N are difficult to use as indicators because they are less reproducible owing to their small range of values and their dependence on how the ROI is set. 22By contrast, a previous study reported that increased LAT1 expression also increased the uptake capacity of BPA by tumors. 29Onishi et al. 30 reported that the overexpression of LAT1 in cancer cells enhanced the therapeutic effect of BNCT in vitro.However, to the best of our knowledge, this is the first report of a correlation between LAT1 expression in human tumor specimens and 18 F-FBPA accumulation in human tumors undergoing 18 F-FBPA PET.

Patients
To assess the biological effects of BNCT accurately, it is essential to consider the intercellular heterogeneity of 10 B distributions. 31 In the present study, no correlation was identified between 18 F-FBPA PET and LAT1 expression and heterogeneity.The following are possible reasons for this: First, as 18 F-FBPA PET is a macroscopic assessment, heterogeneity may not be assessed at the cellular level.Second, the biopsy specimens were assessed for only a part of the tumor, and heterogeneity across the entire tumor may not have been assessed.Both macroscopic and microscopic evaluation by LAT1 expression and 18 F-FBPA PET may solve these problems.Third, the different timings of biopsy and 18 F-FBPA PET/CT imaging may have changed the characteristics of heterogeneity.
This study has two limitations.First, a time lag occurred between specimen collection and 18 F-FBPA PET/ CT imaging in some cases.Second, it was a retrospective study with a small sample size, which may include some bias.Prospective studies investigating the correlation between LAT1 expression and treatment response in patients undergoing BNCT are required.
In conclusion, the evaluation of LAT1 expression by specimen sampling correlated with 18 F-FBPA accumulation on 18 F-FBPA PET/CT imaging; however, in some patients with discrepancies between them, LAT1 expression may be more useful in predicting BNCT treatment response.The LAT1 expression score is a good candidate biomarker for predicting BNCT treatment response and awaits validation by studies investigating its correlation with treatment response in patients undergoing BNCT.

F I G U R E 1
Flowchart of patient inclusion.F I G U R E 2 Examples of the L-type amino acid transporter 1 scoring.

F I G U R E 3
Immunostained images with L-type amino acid transporter 1 (LAT1) antibody and 4-borono-2-18 F-fluoro-phenylalanine ( 18 F-FBPA) positron emission tomography (PET)/computed tomography (CT) images of the same patients.(A-1) Maximum and minimum LAT1 expressions were 3. (A-2) The maximum standardized uptake value (SUV max ) was high (6.78).(B-1) Maximum and minimum LAT1 expressions were 0. (B-2) SUV max was low (2.63).(C) This patient had a discrepancy between the intensity of LAT1 expression and SUV accumulation (Patient A in Table The correlations between the intensity of 4-borono-2-18 F-fluoro-phenylalanine accumulation and L-type amino acid transporter 1 (LAT1) expression.Bolded areas represent factors with p < 0.05.Abbreviations: LAT1, L-type amino acid transporter 1; SUV, standardized uptake value; SUV max , maximum SUV; SUV min , minimum SUV; SUV peak , mean SUV within a 1 cm 3 sphere centered at a single point that is the SUV max .
T A B L E 2

LAT1 max LAT1 min SUV max SUV peak SUV min GTVs (cm 3 ) Pathology Days between sampling and PET
SUV peak , mean SUV within a 1 cm 3 sphere centered at a single point that is the SUV max .